Abstract

Background Elevated lipoprotein(a) (Lp[a]) is associated with aortic stenosis (AS). Oxidized phospholipids (OxPL) are key mediators of calcification in valvular cells and are carried by Lp(a).

Objectives This study sought to determine whether Lp(a) and OxPL are associated with hemodynamic progression of AS and AS-related events.

Methods OxPL on apolipoprotein B-100 (OxPL-apoB), which reflects the biological activity of Lp(a), and Lp(a) levels were measured in 220 patients with mild-to-moderate AS. The primary endpoint was the progression rate of AS, measured by the annualized increase in peak aortic jet velocity in m/s/year by Doppler echocardiography; the secondary endpoint was need for aortic valve replacement and cardiac death during 3.5 ± 1.2 years of follow-up.

Conclusions Elevated Lp(a) and OxPL-apoB levels are associated with faster AS progression and need for aortic valve replacement. These findings support the hypothesis that Lp(a) mediates AS progression through its associated OxPL and provide a rationale for randomized trials of Lp(a)-lowering and OxPL-apoB-lowering therapies in AS. (Aortic Stenosis Progression Observation: Measuring Effects of Rosuvastatin [ASTRONOMER]; NCT00800800)

Aortic stenosis (AS), the most common form of valve disease in the western world, afflicts >1 million individuals in North America (1). Medical therapy to prevent development or reduce progression of AS is currently not available, and aortic valve replacement (AVR) remains the sole option available for the treatment of severe symptomatic AS (2,3).

Mendelian randomization analyses have identified the single-nucleotide polymorphism rs10455872 at the LPA locus as the only genome-wide significant single-nucleotide polymorphism associated with the presence of aortic valve calcification (4). Rs10455872 segregates with smaller numbers of kringle (K)IV2 repeats in the LPA gene, which are inversely associated with lipoprotein(a) (Lp[a]) plasma levels. However, rs10455872 is associated with significantly elevated plasma Lp(a) levels beyond what would be predicted from the relationship of KIV repeats alone (5). Subsequent studies have validated these findings and also reported an association between elevated plasma Lp(a) levels and a higher prevalence of AS and need for AVR in the general population (6,7). It has not yet been established if elevated levels of Lp(a) predict progression of pre-existing AS.

Oxidized phospholipids (OxPL) are proinflammatory and proatherogenic (8). We developed an immunoassay that measures the content of OxPL per apolipoprotein B-100 (OxPL-apoB) and found that Lp(a) was the major lipoprotein carrier of OxPL (9,10). A large body of experimental and clinical studies was generated with this assay to support a pathological role of proinflammatory OxPL in mediating anatomic cardiovascular disease, myocardial infarction, stroke, peripheral arterial disease, and risk of cardiac death (11–13).

Experimental studies have reported the presence of oxidized low-density lipoprotein (LDL), which is enriched in OxPL, in aortic valves explanted from surgery and suggested that it is directly involved in pathophysiological processes leading to AS development (14–17). Recently, the enzyme lipoprotein-associated phospholipase A2 (Lp-PLA2), which uses OxPL as a substrate to generate a free fatty acid and lysophosphatidylcholine (LPC), was shown to be highly expressed in stenotic aortic valves and was involved in the mineralization process of valvular interstitial cells via the production of LPC (18). The plasma level of Lp-PLA2 was also associated with a faster progression rate in patients with mild AS at baseline (19). In addition, experimental studies have identified OxPL as key mediators of osteogenic differentiation and calcification in vascular cells (20). These findings support a role for an Lp(a)/OxPL/LPC pathway in AS development and progression (21).

A unifying hypothesis can be proposed that Lp(a) mediates AS through its content of OxPL. In this analysis of the ASTRONOMER (Aortic Stenosis Progression Observation: Measuring Effects of Rosuvastatin) trial, we tested the hypothesis that Lp(a) and OxPL-apoB are associated with hemodynamic progression of AS and AS-related events.

Clinical and Doppler echocardiographic data were previously described (Online Appendix) (23). The primary echocardiographic parameter to assess AS severity was Vpeak. Aortic valve area was also derived from the primary echocardiographic measurements; because the findings are similar to Vpeak, the results are in the Online Appendix.

Fasting plasma samples were collected and stored at —80°C. Plasma levels of glucose, creatinine, total cholesterol, LDL-C, high-density lipoprotein cholesterol, triglycerides, and apoB were measured using automated techniques standardized with the Canadian reference laboratory. LDL-C was corrected for the cholesterol content in Lp(a) using the following formula: corrected LDL-C = LDL-C – Lp(a) mass in mg/dl × 0.3 (24). OxPL-apoB, OxPL on apolipoprotein(a) (OxPL-apo(a)), and Lp(a) were measured with chemiluminescent immunoassays (Online Appendix) as previously described (10,11,25,26). Forty-nine (18.0%) patients were excluded from the current analysis because of lack of plasma samples, leaving 220 patients (82.0%) for the measurement of OxPL-apoB, OxPL-apo(a), and Lp(a) plasma levels. These patients had similar baseline clinical and Doppler echocardiographic characteristics compared with those without OxPL-apoB, OxPL-apo(a), and Lp(a) measurements. Because OxPL-apoB and OxPL-apo(a) gave very similar results, data from OxPL-apo(a) assay also are in the Online Appendix.

Study outcomes

The primary outcome variable was the progression rate of valve stenosis measured as annualized changes in Vpeak. To account for different follow-up lengths, annualized changes in Vpeak were calculated by dividing the difference between last follow-up and baseline values by the length of follow-up. The secondary outcome was the composite of AVR or cardiac death.

Statistical analysis

Continuous data were expressed as mean ± SD and were tested for normality of distribution and homogeneity of variances with the Shapiro-Wilk and Levene tests, respectively. Lp(a) and OxPL-apoB plasma levels were reported as median (interquartile range). Continuous data were compared with the unpaired Student t test across tertiles of Lp(a) and OxPL-apoB. Categorical data were expressed as percentage and compared across tertiles of Lp(a) and OxPL-apoB with the chi-square or Fisher exact tests as appropriate. Multivariable linear regression analyses were performed to identify the independent predictors of AS progression as annualized progression rates of Vpeak, and results were reported as standardized raw-score regression coefficient (beta coefficient) ± SE. The multivariable model included the following variables: 1) variables with a p value < 0.10 in individual analysis; 2) traditional cardiovascular risk factors; 3) aortic valve phenotype (bicuspid vs. tricuspid); and 4) randomization status (statin vs. placebo) (23). Lp(a) and OxPL-apoB levels were not included in the same model because of the important overlap between tertiles. Univariable and multivariable logistic regression models were performed to identify the risk of being rapid progressors, defined as an annualized progression rate of Vpeak ≥0.20 m/s/year as previously described (27). Results were reported as odds ratios (OR) with 95% confidence intervals (CI). Two-way analyses of variance followed by Tukey post-hoc tests were used to evaluate the effect of Lp(a) or OxPL-apoB in the top tertiles and age, aortic valve phenotype, or randomization status on stenosis progression rate. Two-way analyses of variance for repeated measures followed by Tukey post-hoc tests were used to determine the effect of treatment and time (baseline vs. 1-year follow-up) on Lp(a) and OxPL-apoB levels. Kaplan-Meier curves of the time-to-event data were used to assess the effect of Lp(a) or OxPL-apoB in the top tertile on the composite of AVR or cardiac death. Multivariable Cox proportional hazards models adjusted for age, sex, and baseline AS severity were performed to assess the independent association between Lp(a) or OxPL-apoB in the top tertile and the composite of AVR or cardiac death, and results were reported as hazard ratios with 95% CI. Adjusted Kaplan-Meier curves were generated from the adjusted survival function obtained following Cox models. A p value <0.05 was considered statistically significant.

Results

Population characteristics

Baseline clinical, laboratory, and Doppler echocardiographic characteristics are presented (Table 1) according to top tertile of Lp(a) (>58.5 mg/dl) and OxPL-apoB (>5.50 nM) versus middle and bottom tertiles (Lp[a] ≤58.5 mg/dl and OxPL-apoB ≤5.50 nM). In the overall group, median Lp(a) levels were 29.8 mg/dl (12.1 to 76.1 mg/dl) and median OxPL-apoB levels were 3.47 nM (2.26 to 8.64 nM). Eighty-one (37.0%) patients had Lp(a) levels >50 mg/dl. Plasma levels of Lp(a) and OxPL-apoB were strongly correlated (r = 0.86; p < 0.0001). Only 3 patients in the top tertile of Lp(a) were in the middle or bottom tertiles of OxPL-apoB, and 3 patients in the top tertile of OxPL-apoB were in the middle or bottom tertiles of Lp(a). Patients in top Lp(a) or OxPL-apoB tertiles had similar clinical characteristics compared with patients with lower Lp(a) or OxPL-apoB, but had lower LDL-C corrected for Lp(a) cholesterol. The prevalence of bicuspid aortic valve phenotype and hemodynamic AS severity were similar between tertiles. The AV calcification score assessed by echocardiography was significantly higher in the top OxPL-apoB tertile (Table 1).

Risk of Rapid Calcific Aortic Valve Stenosis Progression According to Plasma Levels of Lp(a) and OxPL-apoB

The analysis of annualized progression rate of aortic valve area and the assessment of the relationship between OxPL-apo(a) and AS progression rate provided consistent results (Online Appendix).

There was a significant interaction between patient age and Lp(a) (p = 0.04) (Figure 1C) and a trend between age and OxPL-apoB (p = 0.07) (Figure 1D) for AS progression. Among patients with age ≤57 years (i.e., median age in the whole cohort), AS progression was 2-fold faster in those in the top Lp(a) tertile compared with the middle and bottom tertiles (+0.26 ± 0.32 m/s/year vs. +0.13 ± 0.17 m/s/year; p = 0.02) (Figure 1C); similar results were obtained comparing the top OxPL-apoB tertile with the middle and bottom tertiles (+0.25 ± 0.32 m/s/year vs. +0.13 ± 0.17 m/s/year; p = 0.05) (Figure 1D). However, in patients age >57 years, AS progression rate did not differ according to Lp(a) or OxPL-apoB (Figures 1C and 1D).

After similar multivariable adjustment used in the overall group except for age and smoking history, both top tertiles of Lp(a) (beta coefficient: 0.35 ± 0.06; p = 0.003) and OxPL-apoB (beta coefficient: 0.35 ± 0.06; p = 0.002) remained independent predictors of faster AS progression rate in patients age ≤57 years. Among those age ≤57 years, patients in the top Lp(a) or OxPL-apoB tertiles were at higher risk of being rapid progressors (all p ≤ 0.01) (Table 2).

In the subset of patients age ≤57 years, results on aortic valve area progression were similar to those from Vpeak progression, and the relationship between OxPL-apo(a) and AS progression rate were similar to those obtained with OxPL-apoB (Online Appendix).

Event-Free Survival as a Function of Lp(a) and OxPL-apoB Plasma Levels

Univariable (A, C, E, and G) and adjusted (B, D, F, and H) Kaplan-Meier curves show the composite endpoint of aortic valve replacement or cardiac death comparing tertiles of Lp(a) (A, B, E, and F) and OxPL-apoB (C, D, G and H) in the whole cohort (A to D) and in patients age ≤57 years (E to H). *HR and 95% confidence interval adjusted for age, sex, and baseline Vpeak; †HR and 95% confidence interval adjusted for sex and baseline Vpeak. Numbers at graph bottoms = number of patients at risk at each follow time. HR = hazard ratio; other abbreviations as in Figure 1.

Effect of rosuvastatin therapy

There was no significant interaction between randomization status and Lp(a) or OxPL-apoB (p > 0.05) (Online Figures 1C and 1D) for AS progression rate. From baseline to 1-year post-randomization, Lp(a) and OxPL-apoB plasma levels increased significantly (+20% and +46%, respectively; p < 0.05) in patients treated with rosuvastatin, whereas they did not change in patients on placebo (+3% and +13%, respectively) (Figures 3A and 3B). OxPL-apoB levels were significantly higher at 1 year in the rosuvastatin arm compared with placebo (Figure 3B).

Change in Lp(a) and OxPL-apoB Plasma Levels From Baseline to 1 Year Post-Randomization

Change in plasma levels of Lp(a) (A) and OxPL-apoB (B) in patients randomized to statin versus those randomized to placebo during the first year of the study (i.e., from baseline to 1 year). p values are for the 2-way ANOVAs. ∗p < 0.05 compared to baseline; †p < 0.05 compared to placebo. Error bars represent the SEM. ANOVA = analysis of variance; other abbreviations as in Figure 1.

Discussion

This ASTRONOMER trial analysis demonstrates for the first time that elevated OxPL-apoB and Lp(a) levels are independently associated with an increased risk of echocardiographically determined AS progression rate. Furthermore, this faster progression rate translated to a higher need for AVR, which was accentuated in younger patients with elevated OxPL-apoB or Lp(a) levels. The risk of Lp(a) as an independent predictor of the progression of AS could be explained by OxPL-apoB (or OxPL-apo[a]) levels, which primarily reflect the content of OxPL on Lp(a) present in both the lipid phase of Lp(a) and covalently bound to the apoA moiety (9,10). These findings support the hypothesis that AS progression and need for AVR are mediated by the OxPL content of Lp(a), and provide a rationale for randomized clinical trials of Lp(a)- and OxPL-apoB-lowering therapies (28) in patients with AS (Central Illustration).

Recent studies reported that genetically elevated plasma levels of Lp(a) are associated with the presence of AS in the general population (4,6,7,21). Thanassoulis et al. (4) reported that genetic variation in the LPA locus (rs10455872) is associated with aortic valve calcification and with incident AS across multiple ethnic groups. In the EPIC (European Prospective Investigation into Cancer)-Norfolk population study, subjects who carried the rs10455872 and with Lp(a) levels >50 mg/dl were at increased risk for AS (6). In the Copenhagen City Heart Study and the Copenhagen General Population Study, elevated Lp(a) levels and corresponding genotypes were associated with increased risk of AS and need for AVR in the general population, with levels >74 mg/dl predicting a 3-fold increased risk (7). The results of these studies suggest that the association between Lp(a) and AS may be causal.

None of the previous studies have examined the association between Lp(a) levels and the progression rate of established AS, which is a more relevant clinical endpoint that tracks closely with AS symptoms and ultimately determines the need for AVR. The current study suggests that approximately 4 of 10 patients with AS have elevated Lp(a) levels. It further documents that patients with elevated levels of Lp(a) and OxPL-apoB have approximately a 2-fold risk of being rapid progressors. Patients with rapid AS progression represent a conundrum in clinical care because the underlying etiology of the progression is often not clear and they require AVR sooner. This study suggests that elevated Lp(a) and/or OxPL-apoB levels may be key contributors to the mechanism of rapid AS progression.

Basic investigations have also provided supporting evidence of a role of lipoprotein disorders, oxidation, and inflammation in AS (29). Oxidized LDL, which is highly enriched in OxPL (30,31), has been implicated in promoting valvular inflammation, ectopic calcification, and bone formation (20), features that are pronounced in severe AS.

Lp(a) binds OxPL (32), and much of the cardiovascular disease risk and events associated with Lp(a) can be explained by its associated OxPL (13,32,33). In some studies, OxPL-apoB has been additive or superior to Lp(a) to predicting angiographically determined coronary artery disease (34) or cardiovascular events (13,33). OxPL are a substrate for Lp-PLA2 to produce LPC, which is highly expressed in the aortic valve and promotes valve mineralization (18). Blood Lp-PLA2 activity is associated with a faster stenosis progression rate in the subset of patients with mild AS (19). Finally, it has been shown that OxPL-apoB and Lp-PLA2 potentiate risk of cardiovascular disease events when combined in risk models (35) and that the risk of Lp(a) and OxPL-apoB in mediating angiographically determined coronary artery disease and events was conditional on the presence of a highly inflammatory interleukin-1 genotype (13). Whether this interaction also plays a role in AS remains to be determined.

The interaction between age and Lp(a) levels observed in the present study was potent, suggesting approximately 4-fold faster AS progression rate in patients age ≤57 years. This suggests that the pathophysiological mechanisms leading to the development and progression of AS may be explained in part by the fact that, like most genetic risk factors, patients have elevated Lp(a) starting at birth and this manifests in disease earlier in life. The changes in the valves consequent to the changes induced by OxPL lead to calcification and other pathological changes, which dominate the natural history early in life. As patients age, other risk factors add to the risk of Lp(a) and OxPL-apoB; consequently, they may lose some of their independent predictive value. Additionally, other mechanisms may be involved in the older population, including increased dysregulation in mineral metabolism, post-menopausal deficiency in estrogen, and other aging processes (36,37).

Although data from certain animal models, Mendelian randomization, and retrospective clinical studies suggest that LDL-C could be an important initiator of AS, randomized clinical trials failed to demonstrate any significant benefit of aggressive LDL-C-lowering therapy with statins in patients with mild-to-moderate AS. This lack of efficacy of statins in AS could be related to the fact that these trials only targeted older, normocholesterolemic patients and/or that the beneficial effects of statins on stenosis progression rate were counterbalanced by side effects including: 1) worsening of insulin resistance and LDL particle phenotype as previously reported in a post-hoc analysis of the ASTRONOMER trial (23); 2) promotion of an osteogenic phenotype by statins (38–40); and 3) increased circulating levels of Lp(a) and OxPL-apoB as documented in the present and previous studies (25). Several hypotheses have recently been proposed to explain the association between statins and increased circulating levels of Lp(a) or OxPL-apoB plasma levels (25). However, further studies are needed to confirm the mechanism underlying this association and its clinical relevance.

Clinical implications

It is well appreciated that the rate of AS progression is a primary determinant of developing symptoms of severe stenosis and the need for AVR (29,41). From a clinical standpoint, the most important and urgent need is to develop a pharmacotherapy that is efficient to slow or block the progression of AS in patients who already have the disease. By demonstrating that elevated Lp(a) and OxPL-apoB levels are associated with a faster stenosis progression, the present study provides an important proof of concept for the design of randomized trials to evaluate novel Lp(a)-lowering strategies in AS (28). Circulating Lp(a) levels are genetically determined, and niacin, mipomersen, or experimental drugs only achieve a 20.0% to 40.0% reduction in Lp(a) and also affect other lipoproteins that would confound clinical trial interpretation. Antisense oligonucleotides targeting Lp(a) are currently in phase II trials and have been shown to reduce Lp(a) by a mean of 78.0% and OxPL-apoB and OxPL-apo(a) levels by a similar amount without affecting other lipoproteins (42). In light of the results of the present study and of previous studies (19), the success of future Lp(a) and/or OxPL-apoB-lowering trials may be increased if younger patients with mild AS are targeted.

Study limitations

The relatively small number of patients in each subgroup raises the potential risk of type I statistical error. The ASTRONOMER trial was not powered to assess clinical events, such as AVR and death. Furthermore, the top tertiles of Lp(a) or OxPL-apoB were associated with the composite of AVR or cardiac death only after adjusting for baseline AS severity. Hence, these results should only be considered hypothesis-generating pending a larger analysis. The association between statin treatment and increased level of Lp(a) should be interpreted with caution given that this is a post-hoc analysis and that there was no significant interaction between Lp(a) or OxPL-apoB plasma levels and statin for AS progression. However, our findings are consistent with previous studies that also reported an association between statins and increased levels of Lp(a) and OxPL-apoB (25). The clinical relevance and potential mechanisms to explain these findings are not elucidated and need to be addressed in further clinical studies.

Conclusions

Elevated Lp(a) and OxPL-apoB plasma levels are associated with faster AS progression rate and need for AVR; this association was accentuated in younger patients. This study provides a strong rationale to test Lp(a)-lowering and/or OxPL-apoB-lowering therapies for reducing AS progression and the need for AVR.

Perspectives

COMPETENCY IN MEDICAL KNOWLEDGE: Plasma levels of Lp(a) and OxPL are associated with more rapid hemodynamic progression of valvular AS and associated clinical events, particularly in younger patients.

TRANSLATIONAL OUTLOOK: Randomized trials are needed to evaluate the efficacy of therapies that lower Lp(a) and/or OxPL-apoB on the progression of AS and need for valve replacement.

Acknowledgments

The authors thank all of the investigators of the ASTRONOMER study (listed in the Online Appendix). They also thank Isabelle Gaboury, Lynda Hoey, Judy Keys, Isabelle Laforest, Isabelle Fortin, and Jocelyn Beauchemin for their help in data collection and management.

Appendix

Appendix

For supplemental Methods and Results sections, figures, and a complete list of investigators, please see the online version of this article.

Footnotes

AstraZeneca and the Canadian Institutes of Health Research (CIHR) funded the ASTRONOMER trial. The present substudy was supported in part by grants MOP-114997 to Dr. Pibarot and MOP-245048 to Dr. Mathieu from the CIHR. Dr. Capoulade is supported by a post-doctoral fellowship grant from the CIHR. Drs. Mathieu, Bossé, and Arsenault are research scholars from the Fonds de recherche Québec–Santé. Dr. Mathieu has a patent application on the use of lipoprotein-associated phospholipase A2 inhibitors in the treatment of aortic stenosis. Dr. Després has served as a speaker for Abbott Laboratories, AstraZeneca, GlaxoSmithKline, Merck, and Pfizer Canada, Inc.; has received research funding from Eli Lilly Canada; and has served on the advisory boards of Abbott Laboratories, Torrent Pharmaceuticals Ltd., and Sanofi. Dr. Pibarot holds the Canada Research Chair in Valvular Heart Diseases, CIHR. Drs. Witztum and Tsimikas are supported by National Institutes of Health (National Heart, Lung, and Blood Institute) grants R01-HL119828, R01-HL093767, P01-HL088093, and P01-HL055798; and are coinventors and receive royalties from patents owned by University of California San Diego on oxidation-specific antibodies and of biomarkers related to oxidized lipoproteins. Dr. Witztum is a consultant to Isis Pharmaceuticals, CymaBay, and Intercept. Dr. Tsimikas currently has a dual appointment at the University of California San Diego and as an employee of Isis Pharmaceuticals. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.

References

(2014) 2014 AHA/ACC guideline for the management of patients with valvular heart disease: executive summary: a report of the American College of Cardiology/American Heart Association task force on practice guidelines. J Am Coll Cardiol63:2438–2488.

(2012) Guidelines on the management of valvular heart disease (version 2012). Joint task force on the management of valvular heart disease of the European Society of Cardiology (ESC); European Association for Cardio-Thoracic Surgery (EACTS). Eur Heart J33:2451–2496.

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